Fine Mapping Of A Genetic Male Sterile Gene Ms And PCR Screening Of The BAC Library In Chinese Cabbage

The discovery of genetic multiple-allele male-sterile material in Chinese Cabbage opened up new way for selection and utilization of nuclear male sterile lines. In this laboratory, the Ms genes of Genetic Multiple-allele Male sterile Gene in Chinese Cabbage had been posited in the A07 linkage group. In this experiment, we used the sterile plant of male sterile dual purpose line AB01 in Chinese Cabbage as female parent, hybridized with the FT of DH lines in Chinese Cabbage. And the sterile plant of filial generation backcross with FT, then constructed the large-scale BC1 fine mapping group. And continue developed the SSR markers linked to the sterile gene Ms more closely in the A07 linkage group. At the same time, constructed the genomic BAC Library in Chinese Cabbage, screened chromosome segments in the location region, that laid the foundation for the cloning of Ms gene, the main results as follows:1. Used the separated groups BC1, obtained the SSR molecular marker LZY6 and RF37 linked to the Ms gene, the genetic distance respectively was 0.45cM and 0.62cM. The genetic distance between the RF37 and the LZY6 was 0.17cM, and the direction of the target gene was determined.2. In this experiment, a total of 51840 clones were included in the BAC library. And the original clone number of single hole was 540. The strain of the library was Escherichia coli DH10B, and the vector was plndigoBAC-5. To identify the quality of the BAC library,9 monoclonal were randomly selected from the library. After shaking bacteria, restriction enzyme digestion and so on to detect the average size of the monoclonal insert fragment was 160KB, genome coverage was 15x, the chance of screening positive monoclonal was over 99.9%. The 9 monoclonal randomly selected all contain insert fragments, the no-load rate was 0%.3. Designed primer SKRF1, get the positive monoclonal SRF by PCR screening for the positive mixing pool, positive hole and positive monoclonal of the BAC library, and the fragment size of SRF was about 140Kb. Sequenced the SRF by next-generation sequencing, spliced into 5 scaffold, predicted coding 37 genes. The 37 predicted genes blast in the Brassica database, only 11 known genes. Homology blast of 37 predict genes in Arabidopsis thaliana (Blastx, E<-10),18 genes can be found homologous gene, which had 12 genes knowns function,6 genes unknown function. The remaining 18 genes could be found to match the published sequences in the Nr database, which had 1 genes knowns function,17 genes unknown function.